Aims: Our overall objective is to understand cellular signaling mechanisms, such as that attributable to the endothelial cytosolic free calcium concentration ([Ca2 plus]i), in lung microvascular barrier regulation properties which we quantify as the hydraulic conductivity (LP). The specific aims are to quantify [Ca2 plus]i in intact lung microvessels for the first time, to determine basal [Ca2 plus]i regulation and the extent of intercellular [Ca2 plus]i signaling; mechanisms by which [CA2 plus]i regulates Lp; the significance of gap junctional communication of [Ca2 plus]i in lung microvessels; effects of microvessel distortions on [Ca2 plus]i, and the distribution of the inositol (1,4,5) triphosphate receptor (IP3R) which is an important determinant of [Ca2 plus]i. Procedures: (1) [Ca2 plus]i quantification. Fluorometric imaging of [Ca2 plus]i-sensitive dyes will be conducted intravitally in single microvessels of the isolated, blood-perfused rat lung, using conventional and confocal microscopy. Amplitude and phase locking of [Ca2 plus]i oscillations will be analyzed in the frequency domain by fast Fourier transforms (FFT). Lp will be quantified by the split drop technique. (2) Distribution of IP3R. Using antibodies and cDNA probes for IP3R isoforms, lung microvessels will be subjected to immunohistochemistry, in situ PCR with in situ hybridization, and electronmicroscopy. The effects of prolonged sepsis and hypoxia on IP3R distribution will be determined. Significance: This proposal addresses the understanding of [Ca2 plus]i regulation in intact lung microvessels because dysregulation of [Ca2 plus]i may be common to many mechanisms that cause microvascular barrier deterioration and lead to pulmonary edema. Moreover, sustained increases of [Ca2 plus]i in pathological conditions, may constitute a potent signal for gene transcription and consequently, lung vascular remodeling. If preliminary data bear out, this research will prove for the first time that [Ca2 plus]i regulation is unique in lung microvessels and occurs by means of intercellular [Ca2 plus)I waves. No previous understanding of such mechanisms exist in lung. These proposed studies are therefore, outstandingly novel and important.